The long range objective of our research is to understand the cellular mechanisms governing neuronal excitability. This proposal focuses on the tyrosine kinase dependent suppression of Kv1.2, a voltage gated potassium channel. Kv1.2 is widely expressed throughout the nervous and cardiovascular systems and its suppression is hypothesized to have a key role in human pathologies ranging from neuronal hyperexcitability associated with seizure and stroke to increased vascular tone associated with hypertension. Despite its importance, the mechanism for Kv1.2 suppression remains almost completely unknown. The actin cytoskeleton appears to be central for Kv1.2 regulation since the actin-regulating proteins RhoA and cortactin bind to Kv1.2 and participate in its suppression by tyrosine kinases. Kv1.2 also undergoes tyrosine phosphorylation and actin cytoskeleton dependent endocytosis. This suggests a model in which the physical mechanism of channel suppression involves tyrosine phosphorylation dependent alteration of Kv1.2 interaction with the actin cytoskeleton leading to channel endocytosis and consequent loss of channel function. The goal of this proposal is to understand the molecular mechanisms by which tyrosine kinases, dynamic actin and the endocytotic machinery converge at Kv1.2 to regulate cellular excitability. To do so, a range of biochemical, molecular biological, immunofluorescence microscopy and electrophysiology methods will be used to address the following specific aims: Specific Aim 1: Determine the role of the actin binding protein cortactin in the regulation of Kv1.2 by testing the hypothesis that cortactin acts as a physical conduit between Kv1.2, the actin cytoskeleton, tyrosine kinases and proteins involved in endocytosis. Specific Aim 2: Determine the mechanisms of Kv1.2 endocytosis by testing the hypothesis that individual tyrosines within the channel have specific and divergent roles in ubiquitin dependent channel endocytosis. Specific Aim 3: Elucidate the role of the small G-protein RhoA in Kv1.2 suppression by testing the hypotheses that RhoA bound to Kv1.2 evokes channel suppression by activating effector proteins known to participate in actin filament reorganization. Collectively these aims explore the novel idea that tyrosine kinase signaling, cytoskeletal physiology and protein trafficking act as coordinated players in the regulation of Kv1.2. Thus, the experiments proposed here will provide fundamentally new perspectives into the mechanisms of ion channel regulation and the processes governing neuronal excitability.